Identification of porcine coaglulation factor VIII domains responsible for high level expression via enhanced secretion
Identification of porcine coaglulation factor VIII domains responsible for high level expression via enhanced secretion
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DOI：
10.1074/jbc.m312451200
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发表时间：
2004-02-20
影响因子：
4.8
通讯作者：
Lollar, P
中科院分区：
文献类型：
--
作者：
Doering, CB;Healey, JF;Lollar, P
凝血因子 VIII 具有指定为 A1-A2-B-ap-A3-C1-C2 的结构域结构。人因子 VIII 在正常血浆中浓度较低，相比之下，使用转基因表达技术在体外和体内产生的水平较低。哺乳动物细胞培养物中 B 结构域缺失的猪因子 VIII 的异源表达显着高于 B 结构域缺失的人或鼠因子 VIII。构建新型杂交人/猪因子 VIII 分子来鉴定赋予高水平表达的猪因子 VIII 结构域。杂交人/猪因子 VIII 构建体含有猪因子 VIII A1 和 ap-A3 结构域，其表达水平与重组猪因子 VIII 相当。仅含有猪 A1 结构域的杂合构建体，其表达水平介于人和猪因子 VIII 之间，而含有猪 ap-A3 结构域的杂合构建体，其表达水平与人因子 VIII 相当。此外，含有猪因子VIII A1和ap-A3结构域序列的杂交鼠/猪因子VIII构建体的表达水平显着高于重组鼠因子VIII。因此，猪A1和ap-A3结构域对于与猪因子VIII相关的高水平表达是必要且充分的。代谢放射性标记实验证明高水平表达可归因于分泌效率的提高。
Blood coagulation factor VIII has a domain structure designated A1-A2-B-ap-A3-C1-C2. Human factor VIII is present at low concentration in normal plasma and, comparably, is produced at low levels in vitro and in vivo using transgenic expression techniques. Heterologous expression of B domain-deleted porcine factor VIII in mammalian cell culture is significantly greater than B domain-deleted human or murine factor VIII. Novel hybrid human/porcine factor VIII molecules were constructed to identify porcine factor VIII domains that confer high level expression. Hybrid human/porcine factor VIII constructs containing the porcine factor VIII A1 and ap-A3 domains expressed at levels comparable with recombinant porcine factor VIII. A hybrid construct containing only the porcine A1 domain expressed at intermediate levels between human and porcine factor VIII, whereas a hybrid construct containing the porcine ap-A3 domain expressed at levels comparable with human factor VIII. Additionally, hybrid murine/porcine factor VIII constructs containing the porcine factor VIII A1 and ap-A3 domain sequences expressed at levels significantly higher than recombinant murine factor VIII. Therefore, the porcine Al and ap-A3 domains are necessary and sufficient for the high level expression associated with porcine factor VIII. Metabolic radiolabeling experiments demonstrated that high level expression was attributable to enhanced secretory efficiency.